RNA silencing of rotavirus gene expression

Abstract

RNA interference (RNAi) is a double-stranded RNA (dsRNA)-triggered mechanism for suppressing gene expression, which is conserved in evolution and has emerged as a powerful tool to study gene function. Rotaviruses, the leading cause of severe diarrhea in young children, are formed by three concentric layers of protein, and a genome composed of 11 segments of dsRNA. Here, we show that the RNAi machinery can be triggered to silence rotavirus gene expression by sequence-specific short interfering RNAs (siRNAs). RNAi is also useful for the study of the virus-cell interactions, through the silencing of cellular genes that are potentially important for the replication of the virus. Interestingly, while the translation of mRNAs is readily stopped by the RNAi machinery, the viral transcripts involved in virus genome replication do not seem to be susceptible to RNAi. Since gene silencing by RNAi is very efficient and specific, this system could become a novel therapeutic approach for rotavirus and other virus infections, once efficient methods for in vivo delivery of siRNAs are developed. Although the use of RNAi as an antiviral therapeutic tool remains to be demonstrated, there is no doubt that this technology will influence drastically the way postgenomic virus research is conducted.

Fig. 1

Replication cycle of rotaviruses. The different steps in the replication cycle of the virus are indicated by numbers—1: attachment of the virion to the cell surface; 2: penetration and uncoating of the virus particle to yield DLPs; 3: primary transcription of the genomic dsRNA; 4: synthesis of viral proteins; 5: assembly of core RIs and negative strand RNA synthesis; 6: assembly of double-layered RIs; 7: secondary transcription from double-layered RIs; 8: secondary, enhanced synthesis of viral proteins; 9: secondary, increased assembly of core RIs and negative strand RNA synthesis; 10: secondary, increased assembly of double-layered RIs; 11: budding of double-layered RIs through the membrane of the endoplasmic reticulum (ER), and acquisition of a membrane envelope; 12: loss of the membrane envelope and generation of mature triple-layered virions.

Fig. 2

The RNAi response lasts up to 7 days. MA104 cells in 48-well plates were transfected with an siRNA directed to VP4 (4), VP7 (7), or lamin A/C (C), and after 8 h the lipofectamine–siRNA mixture was removed and replaced by medium. At the indicated times (days post-transfection, d.p.t.), the cells were infected with rotavirus RRV at an MOI of 1 and 12 h post-infection (p.i.), the cells were harvested and processed for electrophoresis or infectivity assays. (A) Western blot stained with a rabbit polyclonal antibody to rotavirus TLPs. Arrows indicate the positions of VP4, VP6, and VP7. (B) The yield of progeny virus obtained after transfection with the siRNA to VP4 was determined by an immunoperoxidase assay, as described (Lizano et al., 1991). Data are expressed as the percentage of the infectivity obtained when the cells were transfected with the control siRNA to lamin A/C.

Fig. 3

Rotavirus gene expression is efficiently inhibited even if RNAi is induced after viral infection. MA104 cells in 48-well plates were infected for 1 h with rotavirus RRV, at an MOI of 3. At the indicated times post-infection (hours post-infection, h.p.i.), the cells were transfected with a siRNA to VP4 (4), or lam A/C(C) as control, for 8 h. At 12 h.p.i. the cells were labeled with [35S]-methionine for 1 h, lysed, and the proteins separated by gel electrophoresis and analyzed by fluorography (A), or by Western blot using a monoclonal antibody to VP4 (B). The dots in (A) indicate the migration of viral proteins other than VP4.